1. The Field of the Invention
The present invention is directed to a methods and apparatus for amplification of DNA.
2. The Relevant Technology
Molecular biology and associated applications rely heavily on the ability to manufacture large amounts of genetic material from small samples so that one can engage in activities such as the identification of particular genetic material in a sample, the measurement of how much genetic material was present, and generation of enough genetic material for use to serve as a component of further applications.
The most successful tool for this purpose is generally known as the “polymerase chain reaction” (PCR). The PCR process is generally performed in a small reaction vial containing constituents for DNA duplication: the DNA to be duplicated, the four nucleotides which are assembled to form DNA, two different types of synthetic DNA called “primers” (one for each of the complementary strands of DNA), salts, and an enzyme called DNA polymerase. These constituents are often referred to as the PCR “cocktail.”
DNA is double stranded. The PCR process begins by separating the two strands of DNA into individual complementary strands, a step which is generally referred to as “denaturation.” This is typically accomplished by heating the PCR reaction mixture to a temperature of about 94 to about 96 degrees centigrade for a period of time between a few seconds to over a minute in duration.
Once the DNA is separated into single strands, the mixture is cooled to about 45 to about 60 degrees centigrade (typically chosen to be about 5 degrees below the temperature at which the primer will melt) in order to allow a primer to bind to each of the corresponding single strands of DNA in the mixture (this involves providing both “upstream” and “downstream” primers). This step is typically called “annealing.” The annealing step typically takes anywhere from a few seconds up to a few minutes.
Next, the reaction vessel is heated to about 72 to 73 degrees centigrade, a temperature at which DNA polymerase in the reaction mixture acts to build a second strand of DNA onto the single strand by adding nucleic acids onto the primer so as to form a double stranded DNA that is identical to that of the original strand of DNA. This step is generally called “extension.” The extension step generally takes from a few seconds to a couple minutes to complete.
This series of three steps, also sometimes referred to as “stages,” define one “cycle.” Hypothetically, proper completion of a PCR cycle results in doubling the amount of DNA in the reaction vial. Repeating a cycle results in another doubling of the amount of DNA in the reaction vial. Typically, the process is repeated many times, e.g. 10 to 40 times, resulting in a large number of identical pieces of DNA. Performing 20 cycles results in more than a million copies of the original DNA sample. Performing 30 cycles results in more than a billion copies of the original DNA sample.
A “thermocycler” is used to automate the process of moving the reaction vessel between the desired temperatures for the desired period of time. Conventional thermocyclers typically require about three hours to run 30 cycles, due to the amount of time required to accomplish a change in temperature for each PCR step, as well as the time required at each target temperature. It would be of great interest in many situations if one could obtain the benefits of PCR more quickly than this.
More recently, thermocyclers have been made available that omit a separate extension stage, and operate as a two stage thermocycler. The first stage of this two stage system is denaturation, and the second stage is annealing, with extension occurring simultaneously with annealing. Although this approach tends to save some time over a three-stage thermocycler, it would still be advantageous to reduce the time required by conventional thermocyclers.